Dengue virus (DENV) is a mosquito-borne member of the Flavivirus genus that has a global impact on public heath due to its widespread distribution and the ability to cause severe disease in humans. Each year, an estimated 390 million individuals are infected by DENV, with clinical manifestations ranging from a self-limiting acute febrile illness (dengue fever) to a potentially fatal syndrome characterized by plasma leakage and shock (dengue hemorrhagic fever; DHF). Four related serotypes of DENV circulate in nature, each capable of causing the full spectrum of DENV-related disease. Prospective clinical studies clearly demonstrate that sequential infection with two DENV serotypes is associated with a more severe disease course. The number of DHF cases reported has increased dramatically during the past twenty years, and now exceeds 250,000 cases annually. Thus, there is an urgent need for the development of a safe and effective vaccine for all four serotypes of DENV. Neutralizing antibodies play an important role in protection against flavivirus infection. Antibodies have been mapped to all three structural domains of the E protein (DI-DIII) that exhibit varying degrees of neutralization potency and confer protection by multiple effector mechanisms. Eliciting neutralizing antibody is a major goal of vaccine development. Complicating these efforts is a requirement for vaccines to simultaneously elicit protection against four different viruses that while antigenically related, share only some of the antibody-binding determinants thought to contribute to virus neutralization. Thus, antibodies raised against one serotype of DENV may react with virions of another serotype, but often with reduced affinity and functional potency. Paradoxically, antibodies may also play a role in enhancing virus infection and exacerbating disease. Antibody-dependent enhancement of infection (ADE) describes a dramatic increase in infection of Fc-receptor-bearing cells in the presence of sub-neutralizing concentrations of antibody or immune sera. The most direct link between ADE and the clinical outcome of DENV infection comes from investigations of the unusually large number of severe DENV cases following primary infection observed in infants during the first year of life. In a broader context, antibodies elicited by primary infection with one serotype of DENV may bind related viruses introduced during secondary infection with reduced avidity, resulting in engagement of the virion with a stoichiometry that does not permit virus neutralization but can support ADE. The development of an immune response that elicits protective levels of neutralizing antibodies against all four serotypes of virus present in the vaccine is a key factor for reducing the risk of ADE. The development of a protective tetravalent response is complicated by the possibility that all four components of a live attenuated tetravalent vaccine may not be equally immunogenic in the vaccinee. Interference and uneven levels of infectivity among DENV strains in this context has been reported. Understanding the immunogenicity of each component of a tetravalent vaccine is an important aspect of vaccine development and identifying appropriate correlates of protection, particularly because it is presently unclear how many serotype-specific responses will be required for protection from all four serotypes of DENV. However, dissecting the specific contribution of each element of a tetravalent vaccine is technically challenging due to the presence of antibodies that bind cross-reactive determinants shared by different components of the vaccine. A detailed map of the functionally important type-specific and group-reactive epitopes on the E protein is not presently available. Improved methodology that distinguishes and quantifies the functional contribution of each component of a tetravalent vaccine would allow for improvements in estimates of vaccine immunogenicity, a more precise correlate of protection, and a powerful investigational tool to study vaccine success and failure. To that end, we propose to develop a novel approach to measure the functional contributions of serotype-specific antibodies against the backdrop of a highly cross-reactive tetravalent polyclonal response. A panel of reporter virus particles (RVPs) containing mutations that destroy antibody-binding determinants will be used to identify functionally important epitopes that elicit type-specific or cross-reactive antibodies present in the serum of DENV vaccinees. An iterative screening process will guide selection of RVPs that are either no longer sensitive to neutralization by cross-reactive antibodies or type-specific responses. RVPs with these characteristics provide two complementary and specific methods for detecting type-specific responses in polyclonal sera. These tools will then be employed to characterize the epitopes that comprise the neutralizing antibody response in vaccinated or naturally infected humans.